Quantitative Analysis of Epstein-Barr Virus DNA Load in Bone Marrow Transplant Recipients By Using Real-Time PCR

Quantification of Epstein - Barr virus (EBV) in peripheral blood mononuclear cells (PBMNC) of allogenic bone marrow transplant (BMT) recipients is important because EBV-associated posttransplant Lymphoproliferative disease (PTLD) can occur after transplantation due to immunosup-pression therapy. To this end we chose Real-Time PCR using TaqMan probe. For the standard curve, we cloned BALF5 gene of EBV into a plasmid vector. After purification of the EBV-clone and calculation of plasmid copy number, the standard curve was constructed by using serial dilution of the plasmid clone. We were able to detect from 2 to 107 copies per 2×105 PBMNC with wide linear range. The mean EBV DNA copy number was 103.7 copies per 2×105 PBMNC. In this study, No patient of 35 BMT recipients (275 PBMNC samples) developed PTLD during five months follow up post transplant. EBV copy numbers in 22 samples (3 patients) out of 35 BMT recipients were higher than cut off value with symptoms like fever and pulomonary noddes (9%). The virus load in one patient in the last sample obtained was 72400 copies. We detected low levels of EBV DNA in 20 BMT patients (57/1%). Real-Time PCR is useful to measure virus load in PBMNC. Detection of EBV in PBMNC samples may be valuable predictive marker to prognosis PTLD. Further studies need to determine ac-curate viral cut off value for treatment patients at risk for PTLD.